The introduction of genes into plants has resulted in the development of plants having new and useful phenotypes such as pathogen resistance, higher levels of healthier types of oils, novel production of healthful components such as beta-carotene synthesis in rice, etc. An introduced gene is generally a chimeric gene composed of the coding region that confers the desired trait and regulatory sequences. One regulatory sequence is the promoter, which is located 5′ to the coding region. This sequence is involved in regulating the pattern of expression of a coding region 3′ thereof. The promoter sequence binds RNA polymerase complex as well as one or more transcription factors that are involved in producing the RNA transcript of the coding region.
The promoter region of a gene used in plant transformation is most often derived from a different source than is the coding region. It may be from a different gene of the same species of plant, from a different species of plant, from a plant virus, or it may be a composite of different natural and/or synthetic sequences. Properties of the promoter sequence generally determine the pattern of expression for the coding region that is operably linked to the promoter. Promoters with different characteristics of expression have been described. The promoter may confer constitutive expression as in the case of the widely-used cauliflower mosaic virus (CaMV) 35S promoter. The promoter may confer tissue-specific expression as in the case of the seed-specific phaseolin promoter. The promoter may confer a pattern for developmental changes in expression. The promoter may be induced by an applied chemical compound, or by an environmental condition applied to the plant.
The promoter that is used to regulate a particular coding region is determined by the desired expression pattern for that coding region, which itself is determined by the desired resulting phenotype in the plant. For example, herbicide resistance is desired throughout the plant so the 35S promoter is appropriate for expression of a herbicide-resistance gene. A seed-specific promoter is appropriate for changing the oil content of soybean seed. An endosperm-specific promoter is appropriate for changing the starch composition of corn seed. Control of expression of an introduced gene by the promoter is important because it is sometimes detrimental to have expression of an introduced gene in non-target tissues. For example, altering oil composition in cells throughout the plant would not be desirable.
In some cases it is desirable to have expression specifically in the roots of a plant. An example is for expression of coding regions that produce proteins for fighting pests that attack roots such as fungi (such as Sclerotinia sclerotiorum, among others), insects (such as corn rootworm, among others), and nematodes (such as soybean cyst nematodes and root knot nematodes, among others). Other examples might be for traits that would produce a better root system, with improved nutrient uptake or increased strength.
In the case of fighting plant pests, it is also desirable to have a promoter whose expression is induced by plant pathogens. Contact with the pathogen will induce expression of the promoter, such that a pathogen-fighting protein will be produced at a time when it will be effective in defending the plant. A pathogen-induced promoter may also be used to detect contact with a pathogen, for example by expression of a detectable marker, so that the need for application of pesticides can be assessed.
One reported root-specific and pathogen-induced promoter is the promoter from the alfalfa isoflavone reductase gene (U.S. Pat. No. 5,750,399). When transformed into tobacco plants, however, this promoter was also expressed in stem and floral tissues. It also showed much lower inducibility in tobacco cells than in alfalfa cells. Another root-specific gene promoter, the RB7 promoter sequence from tobacco (U.S. Pat. No. 5,459,252), has been shown to be able to direct expression of GUS in the root meristem and immature central cylinder regions (Yamamoto et al. (1991) Plant Cell 3:371–382). A carrot “root-specific” promoter has also been described (U.S. Pat. No. 5,959,176). Use of this promoter directs GUS to the vascular bundles, particularly in vascular bundles in root. However, the carrot “root-specific” promoter directs expression of GUS in the leaves of Arabidopsis plants.
Expression of foreign genes by tissue specific promoters in plants will allow the development of useful traits in plants. Therefore identification of a promoter that will regulate the expression of coding regions in a root-specific and pathogen-inducible manner is desirable.